Purification and Characterization of Glucose 6-Phosphate Dehydrogenase from Sheep Liver

Authors : Vedat TÜRKOĞLU, Sinan ALDEMİR

Pages : 395-402

View : 10 | Download : 4

Publication Date : 0000-00-00

Article Type : Research Paper

Abstract :Glucose 6-phosphate dehydrogenase insert ignore into journalissuearticles values(D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD); was purified from sheep liver by a simple and rapid method. The purification process consisted of two steps: preparation of the homogenate, and 2`, 5`-adenosine diphosphate insert ignore into journalissuearticles values(ADP); Sepharose 4B affinity chromatography. Through the use of these two consecutive steps, the enzyme was purified with a yield of 35.6% and 1,920 fold, having the specific activity of 11.2 enzyme units insert ignore into journalissuearticles values(EU/mg protein);. A KM of 0.176 mM and a V\max of 0.0179 EU/ml were obtained for G6-P, and 0.0194 mM and 0.0223 EU/ml for NADP+. Enzymatic activity was measured spectrophotometrically according to Beutler`s method at 340 nm and optimal pH and assay temperature were determined. By means of a Lineweaver-Burk plot, the inhibitor constant for NADPH was determined to be Ki, 4.707 \pm 0.49 m M and it was shown to inhibit the enzyme in a non-competitive manner. The purification of enzyme was monitored by sodium dodecyl sulfate insert ignore into journalissuearticles values(SDS); polyacrylamide gel electrophoresis insert ignore into journalissuearticles values(SDS-PAGE);. SDS-PAGE showed a single band at \sim 55 kDa for the enzyme and gel filtration chromatography indicated it to be a dimer.
Keywords : Glucose 6 phosphate dehydrogenase, purification, sheep, liver